Functional Analysis Of An ABC Transporter In Sterol Uptake In Mythimna Separata Larvae

Cholesterol is an indispensable nutrient for insects,and has the functions of forming cell membranes,being the synthetic precursor of ecdysone,and participating in intercellular signal transmission.But insects lack the ability to synthesize cholesterol de novo,so they must acquire cholesterol from their food.The content of cholesterol in plants is extremely low,which cannot meet the growth and development needs of herbivorous insects.Therefore,herbivorous insects need to convert phytosterols into cholesterol for utilization.But not all kinds of plant sterols can be converted into cholesterol,and insects have different absorption and utilization efficiency for different sterols.However,there are few studies on the genes of insect sterol uptake and utilization.Therefore,we took the important agricultural pest Mythimna separata（Walker）as the research object to clarify the potential role of an ABC transporter involved in sterol absorption.The results of the study are showed as follows:Fourteen ABCG subfamily transporters were screened from M.separata transcriptome.Phylogenetic analysis of ABCG subfamily proteins from Homo sapiens,Drosophila melanogaster,Helicoverpa armigera,Tribolium castaneum,Diaphorina citri,Plutella xylostella,Zeugodacus cucurbitae,and M.separata was performed.According to the results of orthologs searching and phylogenetic tree,the target gene was found to be Mse1046 in M.separata.Sequence analysis revealed that the orthologs were very conserved among different insect species.The relative expression levels of Mse1046 in egg and the 4^th instar were lower than that in other instars,and Mse1046 was highly expressed in the tissues of gut and Malpighian tubes.Additionally,Mse1046 was significantly up-regulated by the cholestanol treatment compared to the cholesterol treatment.These preliminary results lay the foundation for further functional studies.We used CRISPR/Cas9 technology to knock out Mse1046,and obtained the homozygous mutant strain successfully.Feeding the Mse1046 homozygous mutant strain with cholesterol diet and cholestanol diet,we found that the amount of cholestanol in the Mse1046 homozygous mutant strain fed with cholestanol diet was significantly higher than that in the wild-type.Besides,the amount of cholestanol in wild-type fed with cholestanol diet supplemented with ABCG2 inhibitor Ko143 was significantly higher than that in wild-type fed with cholestanol diet without inhibitor.These results revealed that Mse1046 did have the function of excreting cholestanol.The effects of two different dietary sterol treatments（cholesterol and cholestanol）on insect performance（larval developmental time,pupal mass,pupation rate and eclosion rate）in Mse1046 homozygous mutant strain were analyzed.The results showed that the accumulation of cholestanol in the armyworm can significantly reduce the eclosion rate,but did not affect other performance.In conclusion,this study identified an ABC transporter Mse1046,which plays an important role in sterol uptake in the armyworm larvae.This can greatly enhance our understanding of sterol metabolism in insects and provide a reference for the selection of new targets for armyworm control.